Nitrogen metabolism in the yeast S. cerevisiae is an excellent model system in which to study eucaryotic transcriptional regulation and regulatory networks. This yeast is able to selectively utilize good nitrogen sources in preference to poor ones. This selectivity (designated nitrogen catabolite repression, NCR) is accomplished through regulated operation of four GATA-transcription factors (Gln3p, Gat1p/Nil1p, Dal80p/Uga43p, Deh1p/ GZF3p) that bind to GATA-containing sequences in the promoters of NCR-sensitive genes. Aggregate work from several laboratories, including our own, has shown that when nitrogen is in excess, Gln3p and Gat1p are phosphorylated and excluded from the nucleus. When nitrogen is limiting, these proteins are no longer phosphorylated, become localized to the nucleus, and mediate NCR-sensitive transcription. We propose to continue our studies of the GATA-family regulon, performing experiments that will elucidate the steps and proteins involved in the regulation of Gln3p, Gat1p and Ure2p activities and intracellular localization. We will also investigate the mechanisms by which Mks1p influences operation of the GATA regulon. We have established genome-wide transcription analysis in our lab and propose to identify: new members of the GATA-factor regulon, the ways in which the member genes are regulated, and how they in turn regulate GATA-factor gene expression and activity. Three new GATA-factors, Gat2p, Gat3p and Gat4p, were discovered in our pilot experiment. We propose to identify the functions of Gat2p, which is the GATA-factor most structurally homologous to Gat1p. We will also determine how Gat2p itself is regulated. We are especially interested in the interface between and integration of GATA-factor regulon components with other regulatory systems in the cell. The complexity of even this simple system is daunting, but will yield highly useful information and experience for developing and interpreting studies of far more complicated yeast and mammalian regulatory cascades.